In the field of infertility treatment, the development of in vitro fertilization techniques has enabled many couples to conceive and bear children whose infertility proved intractable to other treatments. In particular, in vitro techniques may be indicated in situations in which the fallopian tubes are irreversibly damaged, or where sperm is deficient in motility or concentration. More recently the indications for invitro fertilization have expanded, and may include endometriosis, immunological infertility, and cases of unexplained prolonged infertility. Since 1978 when the first child conceived from invitro ovum fertilization was born, there have been many advances in technique.
In general, the steps in in vitro fertilization involve induction of ovulation, retrieval of oocytes, the fertilization event, and transfer of the embryo to a receptive uterus. In inducing ovulation, hormonal regimens are adopted which increase the chances of hyperovulation, so that more than one oocyte reaching metaphase II is obtained. Two basic regimens involve administration of either human menopausal gonadotrophin (hMG) alone, or hMG in combination with clomiphene either simultaneously or sequentially. Serum estradiol is monitored to ensure progressive increase in serum levels with simultaneous monitoring by ultrasonography of the size of the follicles.
Lutinizing hormone (LH) levels are carefully monitored, and when follicular diameter reaches about 18 mm, human chorionic gonadotrophin is administered which induces a surge in LH associated with follicular maturation and ovulation. More recently, administration of gonadotrophin releasing hormone agonists (hGnRH-a) has been used to suppress pituitary activity during the two week period prior to administration of chorionic gonadotrophin (CG). This has improved pregnancy rates because there is dramatic reduction in premature LH surges associated with poor mature oocyte development. For a detailed description of the use of hGnRH agonists in controlling hyperovulation, see Lunenfeld, B, "Past Present and Future of Gonadotrophins," in Advances in Assisted Reproductive Technologies, ed. Mashiach et al., Plenum Press, N.Y.: 1990, at 39.
Recovery of oocytes is performed approximately 34 hours after administration of human chorionic gonadotrophin (hCG). Various ultrasound-guided techniques for oocyte aspiration have been developed and laparoscopy is rarely performed in current practice. Typically an ultrasound transducer is placed on the abdomen and a needle is passed through the abdomen and bladder, or alternatively, transvaginally, into the follicle. Maturity of each aspirated egg is estimated by assessing the compactness of the cumulus surrounding the oocyte. Those with a loosely expanded cumulus are deemed mature, and ready for fertilization after an initial 6 hour incubation in culture media. Those oocytes deemed immature are incubated an additional 24 to 30 hours.
Spermatozoa, washed in tissue culture medium to remove seminal fluid, are further incubated in a 5% carbon dioxide atmosphere for approximately 2 hours. During this period, the sperm cells become "capacitated" as demonstrated by hyperkinetic motility. Some 10-50,000 spermatozoa are then placed in the incubation chamber with the oocytes. Fertilized eggs appear with two pronuclei 15-17 hours post-insemination. Uterine or tubal deposition via cannula is usually carried out after further 37 to 72 hour incubation until the embryo attains the four to eight cell stage. For further details of the in vitro fertilization process, see Seibel, et al., New England J. Med., 318: 828 (1988).
In spite of technical improvements at virtually every step of the invitro fertilization process, the success rate remains disappointingly low. After embryo transfer to the uterus, the implantation rate remains at only about 20-25 percent. There is some improvement in pregnancy rate when multiple embryos are transferred. However, this also increases the chance of multiple birth. It is widely thought that the low success rate results from subtle hormonal imbalances which make the embryo and/or uterus more or less receptive to implantation. Adjustment or better control of hormone responses has led to improvement in oocyte recovery and production in every step of the process except for implantation.
In the primate there are several hormonal systems involved in the reproductive cycle which may affect embryo implantation. Gonadotrophin releasing hormone, also referred to as gonadotrophin releasor hormone (GnRH) has a central function in regulating the reproductive process. GnRH synthesis occurs primarily in the arcuate nucleus region of the hypothalamus. It is transported to and released from the median eminence into the hypothalamic/hypophyseal portal system. Pulsatile production of GnRH stimulates release in the gonadotrope region of the anterior pituitary gland, of LH and follicle stimulating hormone (FSH) into the peripheral circulation.
In the ovary, the primordial follicle consists of an oocyte and an outer layer of granulosa cells. In response to FSH a recruited follicle forms gap junctions between the oocyte and the granulosa cell layer. Growth of the follicle progresses as the oocyte enlarges and the zona pellucida forms. Interestingly, the follicular fluid contains an aromatase enzyme system which can convert androgens to estrogen, which in turn increases the FSH receptor content of the follicle. The presence of estrogen and FSH in the follicle is essential for continued follicular growth. Since the dominant follicle has more FSH receptors, it competes favorably for free FSH which is continually declining in concentration due to estrogen suppression. Thus a dominant follicle emerges. The FSH also stimulates formation of LH receptors, and conversely, LH can stimulate production of its own receptors in FSH-primed cells.
A major function of the secreted estrogen is to maintain peripheral threshold concentrations of estradiol required for induction of the LH surge, prepatory to ovulation. LH stimulates reduction division in the oocyte and luteinization of the granulocyte layer. The LH surge is also associated with synthesis of progesterone and prostaglandins within the follicle which enhance the activity of enzymes involved in rupture of the follicular wall.
In the luteal phase, the formation and continued development of the corpus luteum is maintained by continued LH stimulation and by a surge in progesterone production. If pregnancy does not ensue, the corpus luteum rapidly degenerates at 9 to 11 days post-ovulation. If pregnancy ensues, production by the embryonic placenta of CG rescues the corpus luteum, maintaining the pregnancy. Detectable CG levels appear at the peak of corpus luteum development which stimulate steroid synthesis until placental steroidogenesis is well established by the 7th week of gestation.
Recently, Seshagiri, et al., Human Repro., 9: 1300 (1994) discovered that the secretion of CG first by the embryonic trophoblast and subsequently by the placental syncytiotrophoblast cells is preceded by synthesis of GnRH. Immunocytochemical analysis showed that GnRH, detected as early as the pre-hatching blastocyst stage, was later localized exclusively in cytotrophoblasts and not in syncytiotrophoblasts, and may represent an early differentiation event in the primate peri-implantation embryo.